JCB: Article
Published August 20, 2012
PKA and PDE4D3 anchoring to AKAP9
provides distinct regulation of cAMP signals
at the centrosome
Anna Terrin,1 Stefania Monterisi,2 Alessandra Stangherlin,1 Anna Zoccarato,1 Andreas Koschinski,2
Nicoletta C. Surdo,2 Marco Mongillo,3 Akira Sawa,4 Niove E. Jordanides,5 Joanne C. Mountford,5
and Manuela Zaccolo1,2
Institute of Neuroscience and Psychology and 5Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow,
Glasgow, G12 8QQ, Scotland, UK
2
Department of Physiology, Anatomy and Genetics, Oxford University, Oxford OX1 3QX, England, UK
3
Venetian Institute of Molecular Medicine, 35129 Padova, Italy
4
Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, MD 21287
P
revious work has shown that the protein kinase
A (PKA)–regulated phosphodiesterase (PDE) 4D3
binds to A kinase–anchoring proteins (AKAPs). One
such protein, AKAP9, localizes to the centrosome. In this
paper, we investigate whether a PKA–PDE4D3–AKAP9
complex can generate spatial compartmentalization of
cyclic adenosine monophosphate (cAMP) signaling at the
centrosome. Real-time imaging of fluorescence resonance
energy transfer reporters shows that centrosomal PDE4D3
modulated a dynamic microdomain within which cAMP
concentration selectively changed over the cell cycle.
AKAP9-anchored, centrosomal PKA showed a reduced
activation threshold as a consequence of increased autophosphorylation of its regulatory subunit at S114. Finally,
disruption of the centrosomal cAMP microdomain by local
displacement of PDE4D3 impaired cell cycle progression
as a result of accumulation of cells in prophase. Our findings describe a novel mechanism of PKA activity regulation that relies on binding to AKAPs and consequent
modulation of the enzyme activation threshold rather than
on overall changes in cAMP levels. Further, we provide
for the first time direct evidence that control of cell cycle
progression relies on unique regulation of centrosomal
cAMP/PKA signals.
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THE JOURNAL OF CELL BIOLOGY
1
Introduction
The second messenger cAMP mediates the intracellular response to multiple hormones and neurotransmitters and regulates a wide variety of cellular processes, including gene
expression, metabolism, and cell growth and division (Stork
and Schmitt, 2002). cAMP is generated from ATP by adenylyl
cyclases, and phosphodiesterases (PDEs) provide the only
means to degrade cAMP (Conti and Beavo, 2007). Therefore,
PDEs play a key role in the control of cAMP resting levels as
well as in determining the amplitude and duration of cAMP
signals in response to extracellular stimuli (Houslay, 2010).
The main effector of cAMP is PKA, a tetrameric enzyme that
Correspondence to Manuela Zaccolo: [email protected]
Abbreviations used in this paper: AKAP, A kinase–anchoring protein; AKAR,
A kinase activity reporter; D/D, dimerization/docking; ERK, extracellular regulation kinase; FRET, fluorescence resonance energy transfer; IS, in silico; PDE,
phosphodiesterase; RCF, rat cardiac fibroblast; RIAD, RI-anchoring disruptor;
ROI, region of interest.
in its inactive form consists of two catalytic subunits (C) and
one regulatory subunit (R) dimer. Upon binding of cAMP to
the R subunits, the C subunits are released and phosphorylate
downstream targets.
A multitude of different stimuli can generate an increase
in intracellular cAMP, and active PKA C subunits can potentially phosphorylate a large variety of protein targets within the
same cell. However, in order for the cell to execute the appropriate task in response to a specific stimulus, the correct subset of downstream targets must be phosphorylated. To achieve
this, spatial confinement (compartmentalization) of the molecular components of the cAMP signaling pathway is critical
(Zaccolo, 2009). PKA is tethered to subcellular loci via binding
© 2012 Terrin et al. This article is distributed under the terms of an Attribution–
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Supplemental Material can be found at:
/content/suppl/2012/08/15/jcb.201201059.DC1.html
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J. Cell Biol. Vol. 198 No. 4 607–621
www.jcb.org/cgi/doi/10.1083/jcb.201201059
JCB
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Published August 20, 2012
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cytosol. In addition, we show that selective disruption of cAMP/
PKA signaling at the centrosome has unique effects on cell
cycle progression.
Results
To study cAMP signals at the centrosome, we generated a CHO
cell clone that stably expresses a PKA-based FRET sensor,
PKA-GFP (Vaasa et al., 2010). The sensor includes the regulatory type II (RII-CFP) and the catalytic (C-YFP) subunits of
PKA tagged, at their carboxyl termini, with the cyan and the
yellow variants of the GFP, respectively (Fig. 1 A; Zaccolo
et al., 2000). In the absence of cAMP, the sensor subunits RIICFP and C-YFP interact, allowing energy transfer (FRET) from
the donor CFP to the acceptor YFP. In the presence of cAMP,
the RII-CFP and C-YFP subunits dissociate, and FRET is abolished (Fig. 1 A). We have previously reported that PKA-GFP
shows the same cAMP dependence and the same sensitivity to
cAMP and ability to phosphorylate substrate as wild-type PKA
(Mongillo et al., 2004) and interacts with endogenous AKAPs
via the D/D domain of its RII-CFP subunits (Zaccolo and Pozzan,
2002). Expression of PKA-GFP in CHO cells shows a clear
localization of the sensor at the centrosome both in interphase
cells and in mitotic cells (Fig. 1 B) without affecting centrosome morphology (Fig. S1). Centrosomal localization of the
sensor is confirmed by colocalization of RII-CFP with the centrosomal marker -tubulin (Figs. 1 C and S1). Immunostaining
with CTR453, an mAb specific for AKAP450 (Bailly et al.,
1989; Keryer et al., 2003), shows colocalization of PKA-GFP
and AKAP450 at the centrosome (Fig. 1 C). To assess whether
the centrosomal localization of the sensor is mediated by interaction with centrosomal AKAPs, an RFP-tagged RII subunit
was coexpressed with either GFP-tagged SuperAKAP–in silico
(IS) or GFP-tagged RI-anchoring disruptor (RIAD). The peptides RIAD (Carlson et al., 2006) and SuperAKAP-IS (Gold
et al., 2006) compete selectively with the binding of RI and RII
to AKAPs, respectively. As shown in Fig. 1 D, coexpression of
SuperAKAP-IS GFP, but not RIAD GFP, completely abolishes
the centrosomal localization of RII-RFP.
Basal cAMP levels are lower at the
centrosome than in the bulk cytosol in
interphase cells
CHO cells expressing the PKA-GFP sensor show a small but
highly significant difference in the basal CFP/YFP emission intensity ratio (R) at the centrosome as compared with the bulk
cytosol (Fig. 2 A), indicating that in resting, nonstimulated interphase cells, the level of cAMP at the centrosome is lower
than the mean cAMP level in the cytosol.
A similar difference between bulk cytosol and centrosome
was detected by RII_epac (Fig. S2 A), a unimolecular FRET reporter for cAMP carrying at its amino terminus the D/D domain
from the RII subunit of PKA (Di Benedetto et al., 2008) and therefore, similarly to PKA-GFP, able to interact with endogenous
centrosomal AKAPs (Fig. 2 B). The RII_epac reporter detects
a significant difference between cytosolic and centrosomal basal
cAMP levels in several other cell types analyzed, including the
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to A kinase–anchoring proteins (AKAPs). AKAPs anchor PKA
in proximity to its targets via binding to the amino-terminal
dimerization/docking (D/D) domains of PKA R subunits of
an amphipathic helix within the AKAP sequence (Wong and
Scott, 2004). The cAMP signal is also compartmentalized,
with different intracellular subcompartments showing different
concentrations of the second messenger (Zaccolo and Pozzan,
2002). Different subsets of anchored PKA are thus exposed
to different levels of cAMP, resulting in selective activation
and phosphorylation of the appropriate subset of targets (Di
Benedetto et al., 2008). PDEs, a large superfamily of enzymes
comprised of 11 families (PDE1-11) and >30 isozymes, can
also be localized to specific subcellular compartments and,
by locally degrading cAMP, play a key role in the spatial control of cAMP signal propagation (Mongillo et al., 2004). Long
isoforms of the PDE4 family, including PDE4D3, can be
phosphorylated and activated by PKA (Sette and Conti, 1996;
MacKenzie et al., 2002), and members of the PDE4D subfamily
have been shown to interact with several AKAPs, including
AKAP6 (Dodge et al., 2001), AKAP7 (Stefan et al., 2007),
and AKAP9 (Taskén et al., 2001). The presence of PKA and
PDE4D3 within the same macromolecular complex may thus
provide a negative feedback system in which elevated cAMP
concentrations trigger PKA to phosphorylate and activate
PDE4, reducing local cAMP levels and resetting PKA activity
selectively at that site (Dodge et al., 2001).
AKAP9/450/350/CG-NAP (centrosome- and Golgi-localized,
PKN-associated protein; hereafter referred to as AKAP450)
localizes at the centrosome (Schmidt et al., 1999; Takahashi
et al., 1999; Witczak et al., 1999) through a conserved protein
interaction module known as the PACT (pericentrin-AKAP350
centrosomal targeting) domain (Gillingham and Munro, 2000).
Localization of AKAP450 at the centrosome has been shown to
be required for centrosome integrity and centriole duplication
(Keryer et al., 2003).
The centrosome plays a key role in cell cycle progression
and acts as a scaffold for the accumulation and interaction of
different cell cycle regulators (Cuschieri et al., 2007). PKA has
been shown to be involved in many aspects of cell cycle regulation, including centrosome duplication, S phase, G2 arrest,
mitotic spindle formation, exit from M phase, and cytokinesis
(Matyakhina et al., 2002); however, which, if any, of these
functions is regulated by a PKA subset targeted at the centrosome remains to be established. In addition, it is not clear how
cells achieve appropriate control of cell proliferation while continuously being exposed to hormonal fluctuations and, consequently, to changes in intracellular cAMP levels.
In this study, we use real-time imaging and a combination of fluorescence resonance energy transfer (FRET)–based
reporters to explore the hypothesis that anchoring of PKA
and PDE4D3 to AKAP450 provides a structural basis for
selective regulation of cAMP signals at the centrosome. Our
results show, for the first time, that the centrosome is a subcellular compartment undergoing a sophisticated and dynamic
control of cAMP signals and PKA activation that relies on PKA
anchoring to AKAP450 and on the presence of PDE4D3 and
can be independent of overall changes of cAMP levels in the
Published August 20, 2012
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Figure 1. The FRET sensor PKA-GFP localizes to the centrosome via binding to endogenous AKAPs. (A, top) Schematic representation of the cAMP sensor
PKA-GFP. IS indicates the catalytic inhibitory site and autophosphorylation site. Domains A and B indicate the cAMP-binding domains A and B, respectively.
(bottom) Illustration of the interaction of PKA-GFP with an AKAP (in green) and its mechanism of activation upon binding of cAMP. The yellow and blue
halos around YFP and CFP indicate fluorescence emission from the fluorophores upon excitation of CFP at 430 nm. (B) Subcellular distribution of the sensor
in a CHO cell stably expressing PKA-GFP in interphase (left) and mitosis (right). White arrows point to the centrosome and one of the centrioles. (C) CHO
cells stably expressing PKA-GFP and immunostained with a -tubulin–specific antibody (top row) and with the AKAP450-specific antibody CTR453 (middle
row). A negative control (nc) in which the primary antibody is omitted is shown on the bottom row. The signal from the C-YFP component of the sensor is
not shown. Arrows point to the centrosome. (D) CHO cells expressing SuperAKAP-IS-GFP and RIAD-GFP in combination with RII-RFP. Bars, 10 µm.
macrophage cell line RAW264.7, the human neuroblastoma cell
line SH-SY5Y (Biedler et al., 1978), primary human olfactory
neurons (HONs), primary rat cardiac fibroblasts (RCFs; Fig. 2 C),
and the nontransformed cell line RPE1 (Fig. S3 A).
PDE4D3 is responsible for the low basal
cAMP level at the centrosome
In a variety of cell types, including CHO cells (Fig. S4 A) and
RPE1 (Fig. S3 B), PDE4D3 localizes to the centrosome and has
been shown to bind to AKAP450 (Taskén et al., 2001; McCahill
et al., 2005). Therefore, anchoring of PDE4D3 at the centrosome
may explain the observed low cAMP level at this site. In support
of this hypothesis, selective inhibition of PDE4 enzymes with
10 µM rolipram completely abolished the difference in cAMP
between the centrosome and the bulk cytosol, as detected by
both PKA-GFP and RII_epac (Fig. 3, A–C). In contrast, selective inhibition of PDE2 with 10 µM EHNA (erythro-9-(2hydroxy-3-nonyl)adenine; Fig. 3, D and E) or selective inhibition
of PDE3 with 10 µM cilostamide (Fig. 3 F) did not affect the
gradient between centrosome and cytosol. In further support of
a role of PDE4D3 in maintaining a low basal cAMP level at
the centrosome, genetic knockdown of PDE4D using an siRNA
approach and resulting in an almost complete ablation of all
PDE4D isozymes (Fig. S4 B) completely abolished the differences in cAMP levels between cytosol and centrosome (Fig. 3,
G and H), whereas the control oligonucleotide siGLO did not
show any effect (Fig. 3 I). Anchoring of active PDE4D3 at
the centrosome appears to be necessary to maintain low at the
local cAMP concentration, as shown by experiments in which
we used a catalytically inactive mutant of PDE4D3 (dnPDE4D3;
McCahill et al., 2005). When overexpressed, dnPDE4D3 localizes at the centrosome (Fig. S4 C) and is expected to displace
endogenous active PDE4D3 from its centrosomal anchor sites.
Overexpression of dnPDE4D3 in CHO cells completely abolished the difference in cAMP concentration between the centrosome and the cytosol, as detected by the coexpressed PKA-GFP
Unique PKA signaling at the centrosome • Terrin et al.
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(Fig. 3, J and K). In contrast, overexpression of a catalytically
inactive mutant of a different PDE4 isozyme (dnPDE4A4;
Fig. S4 C; McCahill et al., 2005) did not affect the cAMP
gradient between the cytosol and centrosome (Fig. 3 L). Collectively, the aforementioned data strongly indicate that PDE4D3
is responsible for maintaining a microdomain with low cAMP
concentration at the centrosome.
PKA anchored to AKAP450 shows
increased sensitivity to cAMP
Next, we assessed the cAMP response generated in the bulk
cytosol and at the centrosome upon activation of adenylyl cyclases with forskolin, using either the RII_epac or the PKAGFP reporter. Upon application of 25 µM forskolin, we found
that the RII_epac sensor reported an equal FRET change in the
cytosol and at the centrosome (Fig. 4 A), whereas, unexpectedly, the PKA-GFP reporter recorded a significantly higher
signal at the centrosome than in the bulk cytosol (Fig. 4 B).
To explain this discrepancy, we hypothesized that the higher
FRET change recorded at the centrosome by PKA-GFP may
be the consequence of an increased sensitivity to cAMP of the
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JCB • VOLUME 198 • NUMBER 4 • 2012
centrosomal-targeted, PKA-based biosensor. To verify this
hypothesis, we expressed the PKA-GFP sensor in CHO cells
in combination with a fragment of the centrosomal AKAP450
encompassing amino acids 933–1,804 (AKAP450-2; Witczak
et al., 1999) and including the amphipathic helix responsible for binding the RII subunits of PKA. AKAP450-2 lacks
the PACT domain responsible for anchoring of AKAP450 to the
centrosome (Fig. S2 B; Gillingham and Munro, 2000). Thus,
when expressed in cells, AKAP450-2 is a cytosolic polypeptide
(Fig. 4 C) that retains its ability to bind to PKA RII subunits
(Fig. 4 D). The rationale for this experiment is that if anchoring
of PKA to endogenous AKAP450 at the centrosome affects the
kinase sensitivity to cAMP, the same effect should result from
PKA binding to AKAP450-2 in the cytosol. Fig. 4 E shows that
this is the case, and, as reported in Fig. 4 F, the dose-response
curve where FRET change is plotted against increasing concentrations of forskolin shows a shift to the left for cells coexpressing the FRET sensor and AKAP450-2, confirming that a lower
concentration of cAMP is sufficient to dissociate PKA-GFP
when it is bound to AKAP450. The effect of AKAP450-2 on the
sensitivity of PKA-GFP to cAMP is completely abolished in the
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Figure 2. Basal cAMP in interphase cells is lower at the centrosome than in the cytosol. (A) CHO cell stably expressing the PKA-GFP sensor. The middle
image in grayscale is the image acquired at 480 nm upon excitation at 430 nm and shows the subcellular distribution of the sensor. The signal generated
by the C-YFP component of the PKA-GFP sensor is not shown. The FRET signal from the same cell, calculated as a 480/540-nm emission intensity ratio upon
excitation at 430 nm, is shown in pseudocolor. Images on the left show a higher magnification of the centrosomal region. White arrows point to the centrosome. The image on the right shows the mean basal FRET signal measured in the bulk cytosol (cyt) and at the centrosome (centr) of cells stably expressing
PKA-GFP. FRET values are the mean calculated within an ROI drawn to include the entire cytosolic area or the centrosome (an example is provided in Fig.
4, A and B) and are expressed relative to the FRET value measured in the cytosol. n = 34. (B) CHO cells stably expressing the unimolecular sensor RII_epac.
Images are as described in A. n = 31. (C) Sensor distribution and FRET pseudocolor images of RAW264.7 cells, SH-SY5Y cells, primary HONs, and primary
RCFs expressing RII_epac. For each cell type, bottom images show the mean FRET signal in the cytosol and at the centrosome, calculated as described in A.
n ≥ 5. For all experiments, error bars represent SEM. Two-tailed paired t tests were performed (*, P < 0.05; ***, P < 0.001). Bars, 10 µm.
Published August 20, 2012
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Figure 3. The low cAMP compartment at the centrosome depends on centrosomal PDE4D3. (A) Sensor distribution and FRET pseudocolor image of a representative CHO cell stably expressing PKA-GFP and treated with 10 µM rolipram (Rol). Images on the left are a magnification of the centrosomal region.
(B) Mean basal FRET signal calculated in the cytosol (cyt) and at the centrosome (centr) in CHO cells expressing PKA-GFP and treated with the PDE4 inhibitor rolipram (10 µM). n = 44. (C) Mean basal FRET signal calculated in the cytosol and at the centrosome in CHO cells expressing RII_epac and treated
with 10 µM rolipram. n = 25. (D) Sensor distribution and FRET pseudocolor image of representative CHO cells stably expressing PKA-GFP and treated
with the PDE2 inhibitor EHNA (10 µM). (E) Mean basal FRET signal calculated in the cytosol and at the centrosome in the same cells. n = 46. (F) Mean
basal FRET signal calculated in the cytosol and at the centrosome of CHO cells stably expressing PKA-GFP and treated with the PDE3 inhibitor cilostamide
(Cilo; 10 µM). n = 39. (G–I) Sensor distribution and FRET pseudocolor image of a representative CHO cell stably expressing PKA-GFP and in which PDE4D
isoforms have been knocked down by siRNA treatment (G). The mean FRET signal in the cytosol and centrosome in these cells and in cells expressing the
control sequence siGLO are shown in H (n = 40) and I (n = 40), respectively. (J and K) Sensor distribution and FRET pseudocolor image of a representative CHO cell stably expressing PKA-GFP and a catalytically inactive mutant of PDE4D3 (dnPDE4D3); the mean FRET signal in the cytosol and centrosome
(n = 31) is shown in K. (A, D, G, and J) Arrows point to the centrosome. (L) Summary of the basal CFP/YFP ratio values recorded in the cytosol and at the
centrosome of cells expressing a catalytically inactive mutant of PDE4A4 (n = 21). All error bars represent SEM. Two-tailed paired t tests were performed
(*, P < 0.05; ***, P < 0.001). Bars, 10 µm.
presence of SuperAKAP-IS (Fig. 4 G), confirming that this effect depends on the interaction of PKA-GFP with AKAP450-2.
AKAP450-2 has no effect on the FRET change detected by
a variant of the PKA-GFP sensor (PKA-GFP) in which the
D/D domain of the RII subunit has been deleted (Zaccolo and
Pozzan, 2002), thereby resulting in a sensor that cannot bind
to AKAPs (Fig. 4 H), or when a serine to proline substitution
(S1451P) is introduced in AKAP450-2 (mutAKAP450-2) that
disrupts the amphipathic helix and abolishes the ability of
PKA-GFP to bind (Fig. 4 I; Feliciello et al., 2001; Alto et al.,
2003). As an additional control, we measured the FRET change
reported by the sensor RII_epac when coexpressed with the
AKAP450-2 fragment, and we found no difference compared
with the FRET change recorded in the presence of the sensor
Unique PKA signaling at the centrosome • Terrin et al.
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Figure 4. PKA-GFP anchored to AKAP450 shows increased sensitivity to cAMP. (A, left) CHO cell stably expressing the RII_epac sensor. Representative
ROIs within which the ratio values are averaged for bulk cytosol (black line) and centrosome (gray line) are shown. (right) Normalized mean kinetics
(n = 31) of FRET change induced by 25 µM forskolin (frsk) in CHO cells stably expressing RII_epac and recorded in the cytosol (cyt) and at the centrosome
(centr). (B, left) CHO cell stably expressing the PKA-GFP sensor. (right) Normalized mean kinetics of FRET change detected in response to 25 µM forskolin
in CHO cells stably expressing PKA-GFP and recorded in the cytosol and at the centrosome (n = 35). (C) Distribution of the GFP-tagged AKAP450-2 fragment in a representative CHO cell. (D) Western blot analysis of lysates from CHO cells overexpressing the GFP-tagged AKAP450-2 fragment. AKAP450-2
was immunoprecipitated using GFP-Trap beads, and the total lysate (L), unbound fraction (NB), and protein-bound fraction (B) to the GFP-Trap beads were
immunoblotted with anti-RII antibody. Similar results were obtained in three independent experiments. (E) Normalized mean kinetics (n = 25) of FRET
change induced by 25 µM forskolin in CHO cells expressing PKA-GFP in the presence (open circles) or absence (filled circles) of AKAP450-2. (F) Doseresponse curve of FRET change at different concentrations of forskolin in CHO cells expressing PKA-GFP in the presence (open circles) or absence (filled
circles) of AKAP450-2. For each concentration point, n ≥ 10. (G) FRET change induced by 25 µM forskolin in CHO cells expressing PKA-GFP in the presence or absence of AKAP450-2 fragment and SuperAKAP-IS. n ≥ 18. (H) FRET change induced by 25 µM forskolin in CHO cells expressing the deletion
mutant sensor PKA-GFP in the presence or absence of AKAP450-2. n ≥ 23. (I) FRET change induced by 25 µM forskolin in CHO cells expressing PKA-GFP
in the presence or absence of the AKAP450-2 and mutAKAP450-2. n ≥ 14. (J) FRET change induced by 25 µM forskolin in CHO cells expressing the
RII_epac sensor in the presence or absence of the AKAP450-2. n ≥ 34. (K) FRET change induced by 25 µM forskolin in CHO cells expressing PKA-GFP
in the presence or absence of AKAP79 fragment (amino acid 352 to 428). n ≥ 24. (L) FRET change induced by 25 µM forskolin in CHO cells expressing
PKA-GFP in the presence or absence of AKAP149 fragment (amino acid 284 to 385). n ≥ 24. (M) FRET change induced by 25 µM forskolin in CHO cells
expressing PKA-GFP in the presence or absence of the AKAP Rt31 fragment (amino acid 2 to 1,678). n ≥ 17. All error bars are SEM. Two-tailed unpaired
t tests were performed (*, P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001). Bars, 10 µm.
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Figure 5. Binding of PKA to AKAP450-2 increases
PKA activity. (A) FRET change measured in response to
25 µM forskolin in CHO cells expressing AKAR3 in the
presence or absence of AKAP450-2. n = 53. (B) FRET
change measured in response to 25 µM forskolin in CHO
cells expressing AKAR3 in the presence or absence of
AKAP79 fragment. n = 14. A two-tailed paired t test was
performed. (C) Grayscale image acquired at 480 nm
upon excitation at 430 nm and showing the subcellular
distribution of the RII_AKAR3 sensor. The pseudocolor
image shows the FRET signal from the same cell, calculated
as a 540/480-nm emission intensity ratio upon excitation at 430 nm. Images on the left show a higher magnification of the centrosomal region. Arrows point to the
centrosome. Bar, 10 µm. (D) Mean of basal FRET signal
measured in the bulk cytosol (cyt) and at the centrosome
(centr) of cells stably expressing the RII_AKAR3 sensor.
FRET values are expressed relative to the FRET value measured in the cytosol. n = 30. A two-tailed paired t test was
performed. (E) Mean of FRET change elicited by 100 nM
forskolin (FRSK) in cells stably expressing the RII_AKAR3
sensor and recorded in the cytosol and at the centrosome.
All error bars represent SEM. n = 12. Two-tailed unpaired
t tests were performed (*, P < 0.05; **, 0.001 < P < 0.01;
***, P < 0.001).
We found that RII_AKAR3 detects a higher PKA activity at
the centrosome both in resting conditions (Fig. 5 D) and in
response to forskolin stimulation (Fig. 5 E). The aforementioned results confirm that anchoring of PKA to AKAP450
lowers the activation threshold of PKA, resulting in increased
PKA activity at a given cAMP concentration, and show that
in interphase cells, the low cAMP concentration at the centrosome is sufficient to maintain a higher basal phosphorylation
activity of AKAP450-anchored PKA.
Binding of PKA to AKAP450 results
in increased phosphorylation activity
It is well established that autophosphorylation of the RII subunit
at Ser114 (S114; Kim et al., 2006) results in a reduced activation threshold for PKA (Taylor et al., 1990, 2008). Therefore, we
asked whether anchoring of PKA to AKAP450 may favor autophosphorylation of RII at S114. As shown in Fig. 6 (A and B),
we found that phosphorylation at S114 of both endogenous
RII subunits and overexpressed recombinant RII-CFP subunits
was indeed significantly increased in CHO cells overexpressing
AKAP450-2. To further assess whether autophosphorylation of
RII is the mechanism responsible for the higher sensitivity to
cAMP displayed by the PKA subset anchored to AKAP450, we
generated a mutant of the PKA-GFP sensor (mutPKA-GFP) in
which the RII subunit contains a S114A substitution, resulting
in ablation of RII autophosphorylation (Fig. 6 C; Taylor et al.,
1990; Rodríguez-Vilarrupla et al., 2005; Wehrens et al., 2006).
When mutPKA-GFP was overexpressed in combination with
AKAP450-2 and FRET changes measured in the bulk cytosol,
To establish whether anchoring of PKA to AKAP450 affects
PKA-mediated phosphorylation, we measured the activity of
endogenous PKA using the cytosolic FRET-based A kinase
activity reporter (AKAR) 3 (Allen and Zhang, 2006). As summarized in Fig. 5 A, upon challenge with 25 µM forskolin,
cells overexpressing AKAR3 in combination with AKAP450-2
show a significantly higher cytosolic PKA activity than control cells expressing AKAR3 alone. No difference in PKA
activity was observed when AKAR3 was coexpressed with
the AKAP79 (Fig. 5 B). To establish what is the functional
outcome of having at the centrosome a microdomain with low
cAMP concentration but a subset of PKA with higher sensitivity for cAMP, we used a variant of AKAR3 that includes the
D/D domain at its amino terminus (RII_AKAR3; Stangherlin
et al., 2011) and therefore localizes at the centrosome (Fig. 5 C).
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alone (Fig. 4 J). The increased sensitivity to cAMP appears to be
specific for PKA enzymes anchored to AKAP450, as coexpression of PKA-GFP with fragments from Rt31 (Klussmann et al.,
2001), AKAP79 (Herberg et al., 2000), or AKAP149 (Carlson
et al., 2003), all including the RII-binding amphipathic helix,
did not result in a significant difference in the FRET response
compared with control cells expressing the sensor alone (Fig. 4,
K–M). Overall, the aforementioned data show that anchoring
of PKA to AKAP450 results in an increased sensitivity of the
FRET signal to cAMP, which is indicative of a reduced activation threshold of PKA.
Anchoring of PKA to AKAP450 enhances
RII subunit autophosphorylation
Unique PKA signaling at the centrosome • Terrin et al.
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Published August 20, 2012
the FRET change in response to 25 µM forskolin was of the
same amplitude as the change measured in cells expressing the
mutant sensor alone (Fig. 6 D), indicating that autophosphorylation at S114 is necessary for the ability of AKAP450-2 to affect
the activation threshold of PKA. mutPKA-GFP maintains an
intact D/D domain and can therefore anchor to the centrosome
(Fig. 6 E, inset). When CHO cells expressing mutPKA-GFP
were challenged with 25 µM forskolin and the FRET change
measured in the cytosol and at the centrosome in the same cell,
we found no significant difference between the two compartments (Fig. 6 E), confirming that the higher sensitivity to cAMP
of the centrosome-anchored PKA requires autophosphorylation
of RII at S114.
The centrosomal microdomain with low
cAMP is abrogated in mitosis
To investigate the possible functional relevance of the centrosomal microdomain with low cAMP, we sought to establish whether the difference between cytosolic and centrosomal
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cAMP levels changes in different stages of the cell cycle. As
shown in Fig. 7 A, mitotic cells expressing RII_Epac show a
uniform cAMP level at the centrosome and in the bulk cytosol.
The global cAMP concentration in mitotic cells does not appear
to be significantly different from the global cAMP concentration in interphase cells (Fig. 7 B), indicating that a selective increase in cAMP concentration occurs at the centrosome site in
mitosis. Notably, in agreement with the local increase in cAMP
concentration at the centrosome in mitotic cells, PKA phosphorylation activity is further increased selectively at this site,
as detected by the RII_AKAR3 reporter (3.98% ± 0.57 and
6.69% ± 0.4 higher FRET signal in the centrosome compared
with the cytosol in interphase and mitosis, respectively; P =
0.0005; compare Fig. 7 C and Fig. 5 [C and D]). The aforementioned data indicate that the centrosomal cAMP microdomain is
dynamic and is abrogated in mitotic cells.
As the MAPK extracellular regulation kinase (ERK) has
been shown to inhibit PDE4D3 via phosphorylation of its catalytic domain (Baillie et al., 2000), we asked whether mitogenic
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Figure 6. AKAP450-bound PKA is more sensitive to cAMP activation as a result of increased autophosphorylation of RII. (A) Representative Western blot
analysis of total RII and phospho-RII subunits. Lysates from CHO cells overexpressing PKA-GFP in the presence or absence of AKAP450-2 were blotted and
probed for phospho-RII (top blot) and total RII (bottom blot) using specific antibodies. 80 and 53 kD are the expected molecular masses for recombinant
RII-CFP and endogenous RII subunit, respectively. (B) Quantification of endogenous phospho-RII (pS114 RII) and recombinant phospho–RII-CFP (pS114
RII-CFP). Data are the mean of five independent experiments. A two-tailed unpaired t test was performed. (C) Western blot analysis of lysates from CHO
cells expressing the PKA-GFP sensor or a mutant sensor (mutPKA-GFP) containing an S114A mutation in the RII-CFP subunit. Total and phosphor-RII subunits were detected as in A. (D) FRET changes induced by 25 µM forskolin in CHO cells expressing PKA-GFP (filled bars) and mutPKA-GFP (open bars)
in the presence or absence of the AKAP450-2 fragment. n = 28. A two-tailed unpaired t test was performed. (E) Effect of 25 µM forskolin on the FRET
signal detected in the cytosol (cyt) and at the centrosome (centr) of CHO cells expressing PKA-GFP (filled bars) or mutPKA-GFP (open bars). n = 16. (inset)
Distribution of mutPKA-GFP in a representative CHO cell. The arrow points to the centrosome. Bar, 10 µm. All error bars are SEM. Two-tailed paired
t tests were performed (*, P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001).
Published August 20, 2012
Figure 7. The centrosomal microdomain with low cAMP is abrogated in mitosis. (A) Representative mitotic CHO cell stably
expressing the RII_epac sensor. The grayscale image shows the
subcellular distribution of the RII_epac sensor, and the pseudocolor image shows the FRET signal from the same cell. Arrows
point to one of the centrioles. The histogram on the right displays the mean basal FRET signal measured in the bulk cytosol
(cyt) and at the centrioles (centr) in n = 30 cells. FRET values
are expressed relative to the FRET value measured in the cytosol. (B) Comparison of the FRET signal recorded in the bulk
cytosol in interphase (I) and in mitotic (M) CHO cells stably expressing RII_epac. n = 30. A two-tailed unpaired t test was performed. (C) Representative mitotic CHO cell stably expressing
the RII_AKAR3 sensor. Images are as described in A. n = 22.
A two-tailed paired t test was performed. (D) Mean FRET change
measured before and after treatment with 10 nM EGF in serumdepleted CHO cells stably expressing RII_epac. n = 20. All error
bars represent SEM. Two-tailed unpaired t tests were performed
(*, P < 0.05; ***, P < 0.001). Bars, 10 µm.
Local manipulation of cAMP signals at the
centrosome distinctly affects the cell cycle
It has been previously reported that displacement of the endogenous centrosomal AKAP450 and the consequent delocalization of centrosomal PKA type II impair cell cycle progression
(Keryer et al., 2003), indicating that centrosomal PKA may
play an important role in the control of cell division. The data
presented in the previous section suggest that the unique handling of cAMP signals at the centrosome at different stages of
the cell cycle may be important for the regulation of cell cycle
progression. To test this hypothesis, we used flow cytometric
analysis to monitor the effects on the cell cycle of displacing endogenous PDE4D3 with dnPDE4D3, a maneuver that
results in local increase of cAMP at the centrosome (Fig. 3,
J and K; and Fig. 8 E). We found that CHO cells stably expressing
dnPDE4D3 show a significantly higher number of cells in G2/M
and significantly lower number of cells in S phase, suggestive
of a block of the cell cycle in G2/M (Fig. 8 A). Similar results
were found in cells synchronized in S phase before analysis
(Fig. S5). In contrast, and as previously described (Gützkow
et al., 2002), treatment of CHO cells with 25 µM forskolin,
which ensues a global increase in cAMP levels (Fig. 8 E),
results in a block of the cell cycle in G1, with an increased
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stimuli that activate the MAPK pathway could affect the centrosomal cAMP microdomain. As shown in Fig. 7 D, in CHO cells
stably expressing RII_Epac and treated with 10 nM EGF, the
centrosomal microdomain with low cAMP was completely abrogated as a consequence of a selective increase of the FRET
signal at this site.
proportion of cells with 2N DNA content, a significantly
reduced number of cells in S phase, but no change in G2/M
(Fig. 8 B). Overexpression of a catalytically inactive version
of the control enzyme dnPDE4A4 did not show any effect
on the cell cycle (Fig. 8 C). Inhibition of all PDE4 isoforms
with 10 µM rolipram, a treatment that results in increase of
cAMP both at the centrosome and in the cytosol, albeit at a
lower level than elicited by forskolin (Fig. 8 E), did not result
in a detectable effect on cell cycle progression (Fig. 8 D). The
aforementioned findings show that selective local manipulation
of cAMP at the centrosome activates a downstream pathway
with distinct effects on cell division.
Ablation of the centrosomal microdomain
with low cAMP results in accumulation
of cells in prophase
To gain further insight into the mechanism responsible for accumulation of cells in G2/M when the centrosomal cAMP microdomain is perturbed, we generated a stable CHO cell clone
expressing an RFP-tagged histone 2B (H2B-RFP) alone or in
combination with a GFP-tagged dnPDE4D3 or dnPDE4A4.
H2B-RFP labels the chromatin and allows for identification of
different phases of the cell cycle. As illustrated in Fig. 9 A, interphase cells show a homogeneous red fluorescence in the nucleus, cells in prophase can be clearly identified by the presence
of condensed chromatin and an intact nuclear membrane, and
subsequent mitotic phases can be identified by the position of
the chromosomes along the mitotic fuse. Using this approach,
we assigned cells to one of the following categories: interphase,
prophase, metaphase, or anaphase/telophase. We found that
CHO cells coexpressing H2B-RFP and dnPDE4D3 have a
Unique PKA signaling at the centrosome • Terrin et al.
615
Published August 20, 2012
significantly higher number of cells in prophase compared
with CHO cells expressing H2B-RFP alone or H2B-RFP in
combination with dnPDE4A4 (Fig. 9 B).
Discussion
The data reported here strongly support the novel proposal
that the centrosome in interphase is a subcellular compartment in which basal cAMP levels are lower than in the bulk
cytosol as a consequence of centrosomal localization of PDE4D3,
anchoring of PKA to centrosomal AKAP450 lowers the activation threshold of PKA as a consequence of increased auto­
phos­phorylation of AKAP450-anchored RII subunits at S114,
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JCB • VOLUME 198 • NUMBER 4 • 2012
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Figure 8. Displacement of PDE4D3 results in altered cell cycle progression. (A) Quantification of flow cytometry scan analysis of control CHO
cells and CHO cells stably expressing the RFP-tagged and catalytically
inactive mutant of PDE4D3 (dnPDE4D3mRFP). The same analysis was
performed for CHO cells treated with 25 µM forskolin. (B–D) CHO cells
stably expressing the catalytic inactive mutant of PDE4A4 (dnPDE4A4GFP; C) and CHO cells treated with 10 µM rolipram (Rol; D). Histograms
indicate the mean percentages of cells in various phases of the cell cycle.
Data are the mean of at least six independent experiments. FRSK, forskolin. All error bars are SEM. Two-tailed unpaired t tests were performed
(*, P < 0.05; **, 0.05 < P < 0.01; ***, P < 0.001). (E) Effect of overexpression of dnPDE4D3mRFP, 10 µM rolipram, and 25 µM forskolin
and overexpression of dnPDE4A4-GFP on cytosolic (cyt) and centrosomal
(centr) cAMP compared with untreated and unstimulated CHO control
cells (CTRL). All error bars are SEM. Two-tailed unpaired t tests were
performed (*, P < 0.05; ***, P < 0.001).
the centrosomal cAMP microdomain is dynamic and is abrogated in mitosis, possibly via activation of the MAPK pathway,
and manipulation of cAMP at the centrosome via dis­placement
of PDE4D3 uniquely affects cell cycle progression, resulting in
a highly significant increase in the number of cells in G2/M
phase with accumulation of cells selectively in prophase.
These findings demonstrate that local regulation of cAMP
signals at the centrosome is critical for control of cell division.
In addition to a well-established function as a microtubule
organizing center, the centrosome has recently been shown to
play a role in cell cycle control (Doxsey et al., 2005). For example, active maturation-promoting factor, the key initiator of
mitosis, is found at the centrosome during prophase (Jackman
et al., 2003), and studies in which the centrosome was removed
by microsurgical dissection (Hinchcliffe et al., 2001) or laser
ablation (Khodjakov and Rieder, 2001) have provided direct
evidence for a role of the centrosome in cell cycle progression. Of particular note, cell cycle arrest in G1 (Gillingham and
Munro, 2000; Keryer et al., 2003) as well as a block of cyto­
kinesis (Keryer et al., 2003) were observed when AKAP450 and
PKA were selectively displaced from the centrosome, suggesting that a cAMP/PKA signaling module localized at this site
may serve a critical role. cAMP/PKA signaling has been shown
to be involved in many aspects of cell cycle regulation, including centrosome duplication, S phase, G2 arrest, mitotic spindle
formation, exit from M phase, and cytokinesis (Matyakhina
et al., 2002), and it is possible that different cAMP/PKA signal­ing
modules may be responsible for the regulation of specific cell
cycle–related events. In line with this view, our results show
that whereas a global increase in cAMP levels, as generated by
forskolin stimulation, results in an accumulation of cells in G1,
the local increase of cAMP generated by displacing PDE4D3
from the centrosome has a completely different effect, resulting in accumulation of cells in G2/M. Further investigations
will be necessary to identify the specific targets downstream
of AKAP450-anchored PKA. However, in agreement with our
findings, cAMP/PKA-dependent reduction of histone H3 phosphorylation (Rodriguez-Collazo et al., 2008b), an event that
results in disruption of G2 progression in adenocarcinoma
cells, has been shown to require a concentration of cAMP
that is significantly lower than the amount of cAMP necessary for PKA-mediated phosphorylation of cAMP response
element–binding protein (Rodriguez-Collazo et al., 2008a),
suggesting that the pool of PKA responsible for control of G2
progression is more sensitive to cAMP than the pool of PKA
that regulates gene transcription.
The data reported here clearly show that cAMP signals are
uniquely processed at the centrosome, where a high-sensitivity
PKA subset is associated with a PKA-activatable and ERKinhibitable PDE. Our findings are compatible with a model
whereby the lower activation threshold of PKA tethered to
AKAP450 allows for local activation of PKA at a concentration
of cAMP that is insufficient to activate PKA subsets at other
subcellular locations. Mitogenic stimuli selectively increase
cAMP at the centrosome, resulting in further activation of
PKA at this site in the absence of global increase of cAMP levels.
In agreement with this model, overexpression of a catalytically
Published August 20, 2012
Figure 9. Overexpression of dnPDE4D3 results in
accumulation of cells in prophase. (A) Representative
living CHO cells stably expressing dnPDE4A4RFP and the
GFP-tagged H2B imaged in interphase and in different
phases of the mitotic cycle, as apparent from the analysis
of chromosome condensation. Bar, 10 µm. (B) Summary
of the results of seven independent experiments in which
multiple snapshots of living CHO cells expressing either
GFP-tagged H2B and dnPDE4A4RFP or RFP-tagged H2B
and dnPDE4D3GFP were acquired and cells assigned to
the different stages of the cell cycle, as defined in A.
At least 120 mitotic cells were analyzed for each experiment. Data are expressed as the percentage of mitotic
cells that appears to be in prophase. All error bars are
SEM. *, P < 0.05; **, 0.001 < P < 0.01.
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inactive mutant of PDE4D3 in COS1 cells was shown to
result in its PKA-dependent hyperphosphorylation selectively
at the centrosome even at resting levels of cAMP (McCahill
et al., 2005), suggesting that the centrosomal PDE4D3 modulates activation of the local pool of PKA at basal cAMP concentrations. The feedback loop mechanism described here and
involving a high-sensitivity subset of PKA coupled with a
PKA-activatable and ERK-inhibitable PDE at the centrosome
not only allows tight temporal control of centrosomal cAMP
signals but also provides a potential basis for autonomous
regulation of centrosomal cAMP/PKA-dependent events, independently of global increase in cAMP and therefore of Gs
protein–coupled receptor activation.
The functional relevance of the centrosomal cAMP micro­
domain that we have identified is illustrated by the disruption of
cell cycle progression in CHO cells in which cAMP levels are
selectively elevated at the centrosome via overexpression of a
catalytically inactive PDE4D3. We found that in these conditions,
cell cycle progression is disrupted, and cells accumulate in prophase. Of note, we also found that overexpression of dnPDE4D3
is not tolerated in RPE1 cells (Fig. S3), indicating that the effect
of manipulating the centrosomal cAMP microdomain may be
incompatible with cell cycle progression in nontransformed cells.
The data presented here show that during interphase,
although the cAMP level at the centrosome is lower than in
the bulk cytosol, it is sufficient to maintain a tonic activity of
PKA at this site as a consequence of the reduced activation
threshold of the local PKA subset. This finding is in agreement with the established notion that PKA activity is required to maintain cells in interphase (Bombik and Burger,
1973; Lamb et al., 1991). Our analysis shows that in mitosis, there
is a further increase in PKA activity at the centrosome, raising
the question of how PKA activity is tuned temporally to allow
progression from interphase to mitosis. Further studies with
higher temporal resolution will be necessary to dissect cAMP
signals and PKA activity at the centrosome within the same
cell as it progresses from interphase and through mitosis
to establish whether there is a short temporal window within
which PKA activity is reduced to allow the interphase/
mitosis transition. In this context, the activity of phosphatases
may also be critical, as it may counterbalance substrate phosphorylation by a tonically active centrosomal PKA subset.
Our study uncovers a completely novel mechanism of
PKA activity regulation. Such regulation relies on binding of
PKA to AKAP450 and the consequent reduction of the kinase
activation threshold rather than on changes in the level of cAMP
and is therefore effective only at the sites where AKAP450 is
localized. Noncentrosomal splice variants of AKAP450 localize at the sarcolemma of cardiac myocytes in a complex with
the slowly activating potassium channels IKs (Walsh and Kass,
1988) and to NR1 subunits of glutamate receptors at postsynaptic sites in neurons (Lin et al., 1998), and it will be interesting to
establish whether the regulation described here at the centrosome also operates at these other sites. The novel mechanism
we describe defines a new function for AKAPs and introduces a
further level of complexity to the already sophisticated regulation of cAMP/PKA signaling and may have implications that
extend beyond the control of cell cycle progression.
Materials and methods
Reagent
Forskolin (catalog no. F6886), rolipram (catalog no. R6520), cilostamide
(catalog no. C7971), EHNA (catalog no. E114), H-89 (catalog no. B1427),
and EGF (catalog no. E9644) were purchased from Sigma-Aldrich.
Unique PKA signaling at the centrosome • Terrin et al.
617
Published August 20, 2012
Generation of fluorescent chimeras
The CFP-tagged R subunit of PKA (Lissandron et al., 2005) was subcloned into pCDNA3.1/Zeo (+) as an NheI–XbaI fragment. For the generation of mutRII-CFP, the S114A mutation was introduced using the
QuikChange site-directed mutagenesis kit (Agilent Technologies). The
AKAP450-2 fragment (provided by K. Taskén, University of Oslo, Oslo,
Norway) from amino acid 933 to amino acid 1,804 encoded by the
AKAP450 cDNA (DDBJ/EMBL/GenBank accession no. AJ131693;
Witczak et al., 1999) was amplified by PCR and subcloned as an NheI–
BamHI fragment in pcDNA3.1/Hygro (+). For mutAKAP450-2, the S1451P
mutation was introduced using the QuikChange site-directed mutagenesis
kit. The Rt31 fragment from amino acid 2 to amino acid 1,678 (GenBank/
EMBL/DDBJ accession no. AF387102; Klussmann et al., 2001) was PCR
amplified and subcloned as an EcoRI–XhoI insert into pIRES vector (catalog
no. 631605; Takara Bio Inc.). Generation of dnPDE4D3mRFP, the sequence encoding for dnPDE4D3 (McCahill et al., 2005), was amplified
by PCR and subcloned into the BstXI site of the multiple cloning site of
pcDNA3.1/Hygro (+). The monomeric RFP was then inserted as an
XhoI–XbaI fragment in frame at the C terminus of dnPDE4D3. A schematic
of the sensors and AKAP fragments used in this study is shown in Fig. S2.
AKAR3 was a gift from J. Zhang (Johns Hopkins Institute, Baltimore,
MA), AKAP-IS-GFP was a gift from J.D. Scott (Howard Hughes Medical
Institute, University of Washington, Seattle, WA), and RIAD-GFP was a
gift from K. Taskén.
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RT-PCR
Total mRNA was extracted with TRIzol reagent (catalog no. 15596-026;
Invitrogen) from cells transfected with dnPDE4D3mRFP before or after selection with Geneticin. An aliquot of total mRNA was reversed transcribed
with 1 µl SuperScript II Reverse Transcriptase (2,000 U/µl; catalog no.
18064-022; Invitrogen) to generate cDNA. Amplification of the coding regions of dnPDE4D3mRFP was performed by using specific primers annealing on the D484A mutation (McCahill et al., 2005). The primers used are as
follows: 5-GGTAACCGGCCCTTGACTG-3 and 5-GGTTCTTCAGAATATGGTGCACTGTGCAGAT-3 for amplification of PDE4D3 wild type and
5-GGTAACCGGCCCTTGACTG-3 and 5-GGTTCTTCAGAATATGGTGCACTGTGCAGCA-3 for amplification of dnPDE4D3.
FRET imaging
Cells stably or transiently expressing a FRET-based cAMP sensor were imaged 24 h after transfection, as described in Monterisi et al. (2012). In brief,
cells were imaged on an inverted microscope (IX81; Olympus) equipped
with a Plan Apochromat N 60×/1.42 oil immersion objective, a chargecoupled device camera (ORCA-AG; Hamamatsu Photonics), and a custommade beam splitter including the specific set of emission filters for CFP and
YFP acquisition (dichroic mirror 505DCLP, YFP emission of 545 nm, and
CFP emission of 480 nm; Chroma Technology Corp.). During FRET experiments, cells were bathed with 37°C prewarmed PBS. Images were acquired
using CellR software (Olympus) and processed using ImageJ (National
Institutes of Health). FRET changes were measured in different cell compartments by drawing a region of interest (ROI) around a specific compartment
(centrosome or cytosol). FRET changes of all the cAMP sensors were measured as changes in the 480/545-nm fluorescence emission intensities
after background subtraction on excitation at 430 nm. For AKAR3 and
RII_AKAR3 sensors, FRET changes were measured as changes in the ratio
between 545/480-nm fluorescent emission intensities after background
subtraction upon excitation at 430 nm. For dynamic FRET changes, the
kinetic of the 480/545-nm emission intensity ratio is plotted against time,
and the mean FRET response is expressed as the percentage of R/R0, in
which R = R  R0. R0 is the ratio at time = 0 s, and R is the ratio at time =
t seconds. For steady-state (or basal) FRET, 480/545-nm emission intensity
values measured in the cytosol (Rcyt) and at the centrosome (Rcentr) are expressed as normalized values with respect to the basal FRET ratio value
measured in the cytosol. Ratiometric images are displayed in pseudocolor,
according to a user-defined lookup table that assigns a different color to
each ratio value, as indicated.
Western blotting and immunoprecipitation
Untransfected CHO cells or CHO cells stably expressing PKA-GFP
were seeded on 10-cm tissue culture dishes, treated as indicated, and
washed twice with ice-cold D-PBS before cell lysis. Cell lysates were
prepared in lysis buffer containing 25 mM Hepes, pH 7.5, 2.5 mM
EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 10% (vol/vol)
glycerol, 1% (vol/vol) Triton X-100 (catalog no. T8532; Sigma-Aldrich),
and cOmplete EDTA-free protease inhibitor cocktail tablets (catalog no.
11836170001; Roche). AKAP450-GFP was isolated from cell lysates
via immunoprecipitation with GFP-Trap beads (catalog no. gta-100;
ChromoTek) following the manufacturer’s instructions. Protein concentration
was determined using the Bradford protein assay (Bio-Rad Laboratories).
Proteins were separated by gradient gel electrophoresis on NuPAGE Novex
4–12% Bis-Tris gels (Invitrogen) and transferred to polyvinylidene fluoride
membranes (EMD Millipore). Membranes were then blocked either with
protein-free T20 (TBS) blocking buffer (Thermo Fisher Scientific) or 5% (wt/vol)
skimmed milk in TBS-T for 1 h at room temperature. The following antibodies
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Cell culture and transfection
CHO-K1 cells from CHO (Puck et al., 1958) were grown in Ham’s F12
medium (catalog no. 21765-029; Invitrogen) supplemented with 10%
(vol/vol) FBS (catalog no. 10270-106, Invitrogen), 2 mM L-Glutamine
(catalog no. 25030-024; Invitrogen), 100 U/ml penicillin, and 100 µg/ml
streptomycin (catalog no. 15070063; Invitrogen) at 37°C in a humidified atmosphere containing 5% CO2. SH-SY5Y cells from human neuroblastoma (Biedler et al., 1978) were grown in Ham’s F12/DME (1:1;
catalog no. 42430-025; Invitrogen) supplemented with 10% (vol/vol)
FBS, 2 mM L-Glutamine, 1% (vol/vol) nonessential amino acids (catalog no.
11140-035; Invitrogen), 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO 2.
RAW264.7 cells (Raschke et al., 1978) were grown in DME (catalog
no. 41966; Invitrogen) supplemented with 10% (vol/vol) FBS (catalog
no. F9665; Sigma-Aldrich), 2 mM L-Glutamine, 100 U/ml penicillin,
and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Enriched primary cultures of neonatal ventricular heart
fibroblasts (RCF) were obtained from 1–3-d-old Sprague Dawley rats,
as described in Mongillo et al. (2006) . In brief, rats were killed by cervical dislocation, and ventricular tissue was enzymatically digested with
a mixture of collagenase (Roche) and pancreatin (Sigma-Aldrich). The
isolated cell suspension was preplated for 2 h in DME high glucose
(catalog no. 42430025; Invitrogen) supplemented with 20% (vol/vol)
Medium 199 (catalog no. 31150-022; Invitrogen), 5% (vol/vol) horse
serum, 0.5% (vol/vol) newborn calf serum, 2 mM L-Glutamine, 10 U/ml
penicillin, and 10 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. The plastic-adherent nonmyocyte cells
obtained are fibroblasts. These were trypsinized and plated on coverslips for further analysis. HONs were grown in DME/F12 (catalog no.
11330-032; Invitrogen) supplemented with 10% (vol/vol) FBS, 100 U/ml
penicillin, and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. The retinal pigment epithelial RPE1 cells
(Bodnar et al., 1998) were grown in DME/F-12 medium (catalog no.
11320–074; Invitrogen) supplemented with 10% (vol/vol) FBS (catalog
no. 10270-106; Invitrogen), 2 mM L-Glutamine, 100 U/ml penicillin,
and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2.
For transient expression, cells were seeded onto 24-mm glass
coverslips in complete medium and grown for 24 h, as described in
Terrin et al. (2006). Transfections were performed at 50–70% of confluence. All cell types were transfected with TransIT-LT1 reagent (catalog
no. 2300; Mirus Bio LLC) following the supplier’s instructions and using
2–4 µg DNA per coverslip. Experiments were performed 24–48 h
after transfection.
Knockdown of PDE4D was achieved by using an siRNA oligonucleotide targeting the PDE4D gene (125 nM final concentration, 5-GAACUU­
GCCUUGAUGUACA-3 sequence; Thermo Fisher Scientific), as previously
described (Lynch et al., 2005). Control experiments were performed using
siGLO red transfection indicator (125 nM; catalog no. D-001630-02-20;
Thermo Fisher Scientific).
Generation of stable clones
Stable clones expressing the PKA-GFP sensor have been previously described
(Vaasa et al., 2010). In brief, a CHO clone stably expressing RII-CFP
was selected with 300 µg/ml Zeocine and successively used to select a
stable clone expressing the C-YFP subunit by using 800 µg/ml Geneticin
(Promega). CHO clones stably expressing either RII_epac or RII_AKAR3
sensor (Di Benedetto et al., 2008) were obtained by selection with 800 µg/ml
Geneticin. CHO and RPE1 clones stably expressing dnPDE4D3mRFP1
were selected using 700 µg/ml Hygromycin B (Invitrogen). CHO clone stably expressing pcDNA3dnPDE4A4-GFP was selected using 800 µg/ml
Geneticin. Stable clones expressing either the GFP- or the RFP-tagged histone H2B were obtain by selection with 5 µg/ml Blasticidin S (Invitrogen).
In all cases, after 12 d of treatment with the antibiotic, cells were seeded
in a 96-well plate at 0.8 cells/well, and single clones growing in individual wells were selected for further expansion.
Published August 20, 2012
were used to probe the membranes: mouse anti-PKARII (BD), mouse antiPKARII (pS114; BD), goat pan-PDE4D (a gift from M. Houslay), and goat
anti–-tubulin (C-20; Santa Cruz Biotechnology, Inc.). Results, representing
the mean of at least three independent experiments, were normalized to
the amount of -tubulin.
Cells synchronization
G1/S synchrony was obtained by double block with thymidine (SigmaAldrich). In brief, cells were treated with 5 mM thymidine in FBS-free
medium for 16 h, released to cycle in medium supplemented with FBS
for 8 h, and blocked again for an additional 16 h. Cells were allowed
to recover for 24 h in completed medium with or without 10 µM rolipram
before FACS analysis.
Flow cytometry scan analysis
Approximately 106 of cells were treated as indicated and grown in a
T75 flask for 48 h. After 48 h, exponentially growing cells were trypsinized, washed twice with D-PBS, and resuspended in 300 µl D-PBS. 700 µl
of ice-cold 70% (vol/vol) EtOH/PBS was added dropwise, and the
samples were incubated at 4°C for 1 h. After incubation, cells were spun
down, washed with 1 ml D-PBS, resuspended in 250 µl D-PBS containing
5 µl of 10 mg/ml RNAaseA (Sigma-Aldrich), and incubated for 1 h at
37°C. Samples were stained with 5 µl of 1 mg/ml of propidium iodide
and kept in the dark at 4°C until analysis. Flow cytometric analysis was
performed using a FACSCalibur flow cytometer (BD), and data collected
were analyzed with FlowJo software and computed using the DeanJett-Fox model.
Statistics
Data are presented as mean ± SEM. Two-tailed paired and unpaired Student’s t tests were used to determine significance between groups, as indicated. The number of replicates and the type of Student’s t test used are
indicated in the text. Asterisks are used to indicate levels of significance
based on P values: *, P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001.
Online supplemental material
Fig. S1 shows that PKA-GFP localizes at the centrosome and that its over­
expression does not affect the centrosome morphology. Fig. S2 shows a
schematic representation of the RII_epac sensor, AKAP450, and AKAP4502 fragment. A schematic of the interaction between the FRET-based sensors
and AKAP constructs used in this study is also shown. Fig. S3 shows the
localization of the PKA-GFP sensor and PDE4D3 in the nontransformed
cell line RPE1 and also shows analysis of the basal FRET signal at the
centrosome and in the bulk cytosol in the same cell line. Fig. S4 shows
the localization of endogenous PDE4D3 in CHO cells, the efficiency of
its knockdown, and the localization of the overexpressed catalytically
dead PDE4D3 isoform in CHO cells. Fig. S5 shows the FACS analysis
of cell cycle progression in CHO, CHO treated with rolipram, and CHO
stably expressing the catalytically dead PDE4D3 after synchronization in
The authors would like to thank Miles Houslay for dnPDE4D3, dnPDE4A4-GFP,
and goat anti–pan-PDE4D and rabbit anti-PDE4D3 antibodies; John D. Scott
for SuperAKAP-IS-GFP; Kjetil Taskén for AKAP450-2, AKAP79, AKAP149, and
RIAD-GFP; Enno Klussmann (Max-Delbrück-Centrum für Molekulare Medizin,
Berlin, Germany) for Rt31; Jin Zhang for AKAR3; and Guy Keryer for the
CTR453 AKAP450–specific antibody.
This work was supported by the Fondation Leducq (O6 CVD 02),
the British Heart Foundation (PG/07/091/23698), and the National
Science Foundation–National Institutes of Health Collaborative Research in
Computational Neuroscience program (National Institutes of Health R01
AA18060) to M. Zaccolo.
Submitted: 11 January 2012
Accepted: 17 July 2012
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Immunostaining and confocal imaging
Cells transiently or stably expressing the PKA-GFP sensor were washed
three times with ice-cold D-PBS. The centrosome was exposed by treatment
with PHEM solution (45 mM Pipes, 45 mM Hepes, 10 mM EGTA, 5 mM
MgCl2, 1 mM PMSF, and 0.1% (vol/vol) Triton X-100, pH 6.9) for 30 s
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5 min at 20°C, washed twice in D-PBS, and saturated in 3% BSA for 30 min
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